Interleukin-18 (IL-18) mediates ischemic acute tubular necrosis; it has been proved as a rapid, reliable, and affordable test marker for the early detection of acute kidney injury (AKI), but its predictive accuracy varies greatly.MEDLINE and EMBASE, Cochrane Library, Ovid, and Springerlink  were searched for relevant studies (in English) investigating diagnostic accuracy of urine IL-18 to predict AKI in various clinical settings. The text index was increasing or increased urine IL-18 level and the main outcome was the development of AKI, which was primarily based on serum creatinine level . Pooled estimates of diagnostic odds ratio (OR), sensitivity and specificity were calculated. Summary receiver operating characteristic curves were used to calculate the measures of accuracy and Q point value (Q*). Remarkable heterogeneity was explored further by subgroup analysis based on the different clinical settings.We analyzed data from 11 studies of 3 countries covering 2,796 patients. These studies were marked by limitations of threshold and non-threshold effect heterogeneity. Across all settings, the diagnostic OR for urine IL-18 level to predict AKI was 5.11 [95 % confidence interval (CI) 3.22–8.12], with sensitivity and specificity respectively at 0.51 and 0.79. The area under the ROC curve of urine IL-18 level to predict AKI was 0.77 (95 % CI 0.71–0.83). Subgroup analysis showed that urine IL-18 level in pediatric patients (<18 years) and early AKI predictive time (<12 h) were more effective in predicting AKI, with diagnostic ORs of 7.51 (2.99–18.88), 8.18 (2.19–30.51), respectively.Urine IL-18 holds promise as a biomarker in the prediction of AKI but has only moderate diagnostic value. As an abrupt or rapid decline in renal filtration function, acute kidney injury (AKI) is a common condition associated with significant morbidity and mortality. In critically ill patients, those with AKI usually present a worse clinical outcome than their non-AKI counterparts . AlthougInterleukin-18 (IL-18), a member of the IL-1 family of cytokines, is synthesized as an inactive 23-kDa precursor by several tissues including monocytes, macrophages, and proximal tubular epithelial cells, and is processed into an active 18.3 kDa cytokine by caspase-1  as a medA computer-based search was performed in MEDLINE, EMBASE, Cochrane Library, Ovid, and Springerlink databases, from inception until November 15, 2013, to identify potentially relevant articles. The search strategy consisted of terms related to AKI  combined with the term “interleukin-18/interleukin 18/IL-18”. To ensure all articles were located, a secondary search of the articles already retrieved was undertaken, reviewing their reference lists. The preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines for the conduct of meta-analyses were followed [The titles and abstracts were examined independently by two of the authors (X. L. and J. Y.) to ascertain their relevance and inclusion for further analysis. Any disagreements were settled by consensus using a third opinion (Y. Z.). Inclusion criteria were that studies investigated the diagnostic accuracy of urinary IL-18 to predict AKI and had data which could be extracted into a 2 × 2 table or complete data which could be obtained from the corresponding author by e-mail. The search was restricted to human subjects. Only papers in English were eligible.The following variables were recorded or recalculated: study population, sample size, age, sex, baseline IL-18 level, cutoff value for urine IL-18, AKI definition, number of patients who developed AKI, timing of obtaining the specimen, specificity, sensitivity, and area under the ROC curve (AUROC) with 95 % confidence interval (CI). Data for IL-18 sample storage and detection method were also collected. Two reviewers independently excluded articles on the basis of the title and abstract following a custom-made standardized table. The true-positive, true-negative, false-positive, and false-negative results of each included study were quantified. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool [The meta-analysis was conducted using sROC curve as described by Rosman  and the Of 179 primary articles searched, 156 were excluded on the basis of abstract and/or title because they were laboratory studies, genetic studies, review/comment articles, animal studies, or irrelevant to the current analysis; 3 were excluded because non-English articles. Of the 20 remaining articles, 5 studies with accurate diagnostic values of IL-18 missing and 3 studies lacking effective data (though we had contacted the corresponding authors to request the missing information) were excluded. As a result, 11 studies  met the Characteristics of the included trials are shown in Table Table As the respective number of true-positive, false-positive, true-negative, and false-negative results was not provided by the studies, these indexes were calculated from available sensitivity, specificity, and sample size values. Results are reported in Table The distribution of accurate estimator in sROC curve floor plan and the Spearman correlation coefficient  of logarithmic sensitivity and logarithm (1-specificity) showed that there was a threshold effect in different studies Fig. ; Table 4Figure Subgroup analysis Table  showed tThe current study presents a meta-analysis of urine IL-18 for predicting AKI development via the sROC analysis approach, both overall and across a range of subgroups. Studies committed to direct comparisons between urine IL-18 and the “gold standard” sCr were included. The finding derived is consistent with previous studies, which lends more force to the use of urine IL-18 as a marker of AKI in clinical practice. The results of this meta-analysis indicate that urine IL-18 had a pooled diagnostic OR of 5.11 and the estimated area under the curve (AUC) of the mean ROC plot was 0.77 (Q = 0.71), with a high heterogeneity in pooled sensitivity and specificity. This suggests that urine IL-18 has a higher chance of early detecting an AKI compared to sCr, but the application of this biomarker in the diagnosis of AKI should be limited to a certain range in the light of limitations intrinsic to such studies. Since pooled results may increase statistical power and lead to more precise estimates of a treatment effect and the pooled random or fixed effect models reflect the between-study variance , this maDue to the presence of a threshold effect, we used sROC curve fitting, the area under ROC and Q index, to merge the data. In the studies included, cutoff values were expressed in two types, urinary concentration or ratio of urine IL-18 and creatinine concentration, and identified as varying across studies (11.25–125 pg/ml). Such differences can be explained by: (1) use of different reagents in these studies though the assay methods were enzyme-linked immunosorbent assay (ELISA); and (2) differences in clinical settings and study population. Therefore, it might be necessary for each center using urine IL-18 level for early AKI diagnosis to define a specific reference range and cutoff value for each clinical setting.In addition, a significant non-threshold effect heterogeneity exists across these studies. Since these studies were based on different institutions across the world, heterogeneity was inevitable concerning differences in AKI definitions, AKI settings, times for obtaining specimen, and experimental groups admitted to assess the predictive value of urine IL-18. We attempted to use meta-regression to explore the sources of heterogeneity, but failed. So subgroup analysis was performed and proved that urine IL-18 level in pediatric patients and the early AKI predictive time group (<12 h) was more effective in predicting AKI, which might principally account for the heterogeneity. Nevertheless, as the kidney undergoes growth and maturation in neonates who manifest different physiological states due to the abrupt changes at birth, many risk factors are correlated with AKI and the timing of kidney injury remains unknown . The metUsing sCr level as the “gold standard” of diagnosis of AKI is another limitation of such studies because sCr is not an ideal marker of early loss of glomerular filtration or kidney injury . The besAnother limitation lies in IL-18 itself. Cross-sectional studies indicate that urinary IL-18 levels are markedly elevated in patients with acute tubular necrosis compared with healthy controls and a variety of other renal pathologies, including urinary tract infection, chronic renal insufficiency, and pre-renal azotemia . TherefoGenerally speaking, the cost of a single creatinine test is less than a dollar, while that of IL-18 is five times higher. In the course of AKI, biochemical indices need to be monitored and detected many times; given the modest clinical value of IL-18 and its high cost, serum creatinine concentration and change might still be a good indicator rather than IL-18.In conclusion, this meta-analysis included more than 2,700 patients from 11 studies. The result shows that urine IL-18 level is of diagnostic value for AKI. The diagnostic accuracy for AKI tends to be more effective in pediatric patients and early AKI predictive time. To improve the diagnostic value of urine IL-18, more appropriately designed investigations  should be performed.


Although psychotherapy is considered the treatment of choice for patients with personality disorders (PDs), there is no consensus about the optimal level of care for this group of patients. This study reports the results from the 6-year follow-up of the Ullevål Personality Project (UPP), a randomized clinical trial comparing outpatient individual psychotherapy with a long-term step-down treatment program that included a short-term day hospital treatment followed by combined group and individual psychotherapy.The UPP included 113 patients with PDs. Outcome was evaluated after 8 months, 18 months, 3 years and 6 years and was based on a wide range of clinical measures, such as psychosocial functioning, interpersonal problems, symptom severity, and axis I and II diagnoses.At the 6-year follow-up, there were no statistically significant differences in outcome between the treatment groups. Effect sizes ranged from medium to large for all outcome variables in both treatment arms. However, patients in the outpatient group had a marked decline in psychosocial functioning during the period between the 3- and 6-year follow-ups; while psychosocial functioning continued to improve in the step-down group during the same period. This difference between groups was statistically significant.The findings suggest that both hospital-based long-term step-down treatment and long-term outpatient individual psychotherapy may improve symptoms and psychosocial functioning in poorly functioning PD patients. Social and interpersonal functioning continued to improve in the step-down group during the post-treatment phase, indicating that longer-term changes were stimulated during treatment.NCT00378248. Although psychotherapy is a cornerstone in the treatment of personality disorders (PDs), there is little empirical evidence regarding how different levels of psychotherapeutic care may affect treatment outcome -3. GundeIn Europe in particular, treatment models have been developed that combine various levels of care for patients with PDs, known as step-down programs. Such programs consist of an initial intensive treatment in a day hospital or inpatient setting, followed by outpatient psychotherapy with a corresponding reduction in treatment intensity. Existing step-down programs tend to emphasize various group therapies or the combination of individual and group psychotherapy, and the treatment length is typically long-term ,11. ManyDespite the assumption that patients with severe PDs might benefit from long-term treatment, there have been few randomized studies of therapies lasting more than 1 year for patients with severe PDs ,19,20. AThe Ullevål Personality Project (UPP) is a randomized clinical trial (RCT) designed to investigate the effect of two treatment conditions: (1) a step-down program comprising short-term day hospital treatment followed by a combination of long-term group and individual psychotherapy for a maximum of 4 years, or (2) outpatient individual psychotherapy. The results of the 8 month, 18 month and 3 year evaluation have been reported previously -28. The This study aimed to compare long-term outcomes in the two treatment groups using a wide range of clinical measures. More specifically, we addressed three research questions:1. Are there differences in overall clinical course, from baseline to 6 years follow-up on the primary outcome variables, i.e., psychosocial functioning, interpersonal problems, symptom distress, and quality of life?2. Are there any differences in patterns of clinical course, on the primary outcome variables between the 3- and 6-year follow-ups?3. Are there any differences in secondary outcome variables at 6-year follow-up, i.e., diagnostic status, self-harm/suicidality, vocational functioning, and use of health care services?The UPP was conducted at the Department of Personality Psychiatry (DPP) at Oslo University Hospital. The State Health Insurance Fund covered the expenses for both treatment conditions. Patients were evaluated before treatment, and after 8 months, 18 months, 3 years, and 6 years. All patients received optional psychopharmacological consultations with a psychiatrist as part of the follow-up evaluations. The staff at the DPP conducted the initial clinical and diagnostic evaluation, while PhD students and research assistants performed the follow-up interviews and diagnostic evaluations. Written informed consent was obtained from participants after they were provided with a description of the study. The Data Inspectorate and Regional Ethics Committee in Norway approved the project.Two hundred and fifty patients were referred to the DPP at Oslo University Hospital Ullevål during the intake period. Only patients with a diagnosis of PD were included. Exclusion criteria were schizotypal PD, antisocial PD, ongoing alcohol or drug dependence, psychotic disorders, bipolar I disorder, untreated attention deficit hyperactivity disorder (adult type), pervasive developmental disorder , organic syndromes, and being homeless. One hundred and thirty-three patients were excluded because they had conditions listed in the exclusion criteria, did not have any PD, or did not attend the baseline evaluation. The patients’ pre-treatment demographics and clinical characteristics are reported in Table Figure The step-down day hospital treatment program consisted of an initial 18-week day hospital treatment phase, with a combination of psychodynamic and cognitive-behavioral group therapies for 3 to 4 days each week. The written treatment guidelines adhered to relational psychotherapy, with references to group analysis, self-psychology, and mentalization. After the initial day hospital treatment, the patients continued with outpatient combined psychotherapy. The outpatient treatment consisted of weekly group therapy (1.5 hours) for a maximum of 4 years, combined with weekly individual therapy for a maximum of 2.5 years. Fourteen percent of the patients dropped out of the step-down treatment before starting the combined outpatient psychotherapy. The average duration of treatments in the step-down group was 31 months (SD = 16), and the average number of outpatient consultations  was 106 (SD = 76.0). The mean interval between the end of study therapy and the 6-year follow-up analysis (post-treatment phase) was 43 months (SD = 18).The outpatient treatment consisted of different individual psychotherapies conducted mainly by therapists in private practice. The therapists were instructed to treat the patients according to their own preferred method and practice. The researchers gave no instructions to the therapists regarding the duration or intensity of psychotherapy, nor did they interfere with any treatment decisions. The average duration of treatment in the outpatient group was 24 months (SD = 20), and the average number of consultations was 56 (SD = 56.7). The mean interval between the end of study therapy and the 6-year follow-up analysis (post-treatment phase) was 47 months (SD = 20) in the outpatient treatment.To recruit individual therapists to the study, letters were sent to all therapists in Oslo with a contract with the State Health Insurance Fund, as well as professionals working in public mental health outpatient clinics. The therapists were assigned to one of the two treatment arms according to their own preferences, and signed a formal treatment contract with the project.The 10 group therapists in the day hospital treatment were regular staff from the DPP . Seven of the 10 therapists had 5-years of training in group analysis. The mean age of the therapists was 48 years (SD = 9) and 80% were female. These therapists also conducted the outpatient group therapy.The individual therapists (n = 30) involved in the follow-up outpatient treatment included 16 psychologists, 12 psychiatrists, and two psychiatric nurses. Seven were recruited from the regular staff at the DPP. The therapists treated between one and three patients each. The mean age of the individual therapists was 50 years (SD = 9) and 57% were female. Their mean work experience as psychotherapists was 16 years (SD = 8). They were generally very satisfied with their work as therapists; when asked to rate their satisfaction on a scale ranging from 0 (no satisfaction) to 5 (very satisfied), the mean answer was 4.2 (SD = 0.6).Thirty-two external therapists were recruited to provide outpatient treatment . Each therapist treated one to three patients. The mean age of these therapists was 55 years (SD = 8) and 41% were female. The mean work experience as psychotherapists was 20 years (SD = 8). The majority of these therapists practiced psychodynamic theoretical oriented therapy. At the same time, they reported being influenced by therapeutic theory and principles from other therapy models. They reported a mean of 4.0 (SD = 0.6) on the satisfaction scale. The individual therapists in the outpatient treatment arm were somewhat more experienced than the individual therapists in the step-down treatment arm (p < 0.05). The percentage of men was somewhat higher among the therapists in the outpatient group than among the individual therapists in the step-down group; however, this difference did not reach statistical significance.Psychosocial functioning was rated using the Global Assessment of Functioning Scale (GAF) and the Work and Social Adjustment Scale (WSAS) . The GAFSelf-reported interpersonal problems were assessed by the Circumplex of Interpersonal Problems (CIP) , a 48-itSelf-reported symptom distress was measured by the Symptom Checklist, SCL-90-R . The SCLAxis I and II diagnoses were based on the Mini International Neuropsychiatric Interview (MINI)  and the Incidents of self-harm and suicide attempts were self-reported by patients and then confirmed during the research interviews. At baseline and at the 6-year follow-up, the patients were asked to report self-harm and suicide attempts during the last 12 months. When assessed at the 8-month, 18-month, and 3-year follow-ups, patients were asked to report incidents since the last follow-up, as well as suicidal thoughts during the last seven days.t-test (two-tailed) and chi-squared statistics. For the analysis of vocational functioning and the estimates of health care use, the normal distribution range was not fulfilled and non-parametric tests were used. Within-group pre-post effect sizes were computed using Cohen’s d, with pooled pre- and post-SD adjustment for sample size. All analyses were performed using SPSS .All results were analyzed using an intention-to-treat approach based on treatment assignments. Longitudinal analysis was used to assess change over time and relate the changes to covariates, particularly treatment group assignment. We used a linear mixed model (LMM) for the continuous outcomes, with maximum likelihood as the method of estimation. Piecewise linear splines, with one knot at 3-years for all outcomes, were used to describe the main features in the observed outcomes. Separate random intercepts and slopes were included when proven to enhance the model fit. The parameters of main interest were the fixed effect interaction terms between groups and times, prior to and following the knot, describing whether the patients in the two groups changed differently across the observation period. Assumptions about withdrawal from follow-up evaluations  and selection bias follow descriptions in previous reports . Althougd) was 1.07 (range: 0.56-1.92) in the step-down group from baseline to 6 years and 0.87 (range: 0.66-1.10) in the outpatient group.According to the LMM analyses, there were no statistically significant differences between treatment groups at the 6-year follow-up for the primary outcome variables. The p-value of the different outcomes between treatment conditions were: GAF p = 0.52), WSAS (p = 0.47), CIP (p = 0.96), GSI (p = 0.38), BDI (p = 0.47), and QoL (p = 0.27). Both groups improved on all outcome variables between baseline and 6 years. The means and SDs for the main outcome measures, as well as the within-group effect sizes are shown in Table , WSAS  axis I diagnoses, versus 2.3 (SD = 2) in the outpatient group. Thirty percent of the participants at the 6-year follow-up had been diagnosed with at least one PD. The mean number of PDs was 0.45 (SD = 0.75) in the step-down group and 0.31 (SD = 0.59) in the outpatient group. The mean number of PD criteria met was 5.8 (SD = 6.5) in the step-down group and 5.6 (SD = 5.6) in the outpatient group.At the 6-year follow-up, 10% of patients in the step-down group and 12.5% of patients in the outpatient group reported self-harming behavior in the last 12 months. Twenty-one percent of patients in both treatment arms reported suicidal thoughts in the past 7 days. Forty percent of patients in the step-down group and 41% of patients in the outpatient group reported suicidal thoughts in the past 12 months. One patient in the step-down group reported a suicide attempt during the past 12 months, while no suicide attempts were reported in the outpatient group. One patient in the step-down group committed suicide 9 months after withdrawing from the day hospital treatment. According to the Norwegian national registration office, two more patients (from the outpatient treatment arm) had died. The causes of these patients’ deaths are unknown.At the 6-year evaluation, 61% of participants had worked or studied at some point during the past year: 67% in the step-down group and 54% in the outpatient group. The median number of months spent working or studying was 7 (range: 0–12) in the step-down group and 2 (range: 0–12) in the outpatient group. This difference was not statistically significant. However, 63% of patients in the step-down group and 34% of patients in the outpatient group had a GAF score >60 (p = 0.018). The use of health care services, like contact with a general practitioner and current outpatient treatment, did not differ significantly between groups (Table First, there were no statistically significant differences between the treatment groups regarding the primary outcome variables from baseline to the 6-year follow-up. Both groups exhibited clinical improvement during the 6-year period. The effect sizes were mainly in the large range for both treatment groups; although, there was a tendency towards larger effect sizes in the step-down treatment arm. Second, patients in the outpatient group had a marked decline in psychosocial functioning during the period between the 3- and 6-year follow-ups; while, psychosocial functioning continued to improve during the same period in the step-down group. This effect was statistically significant. Finally, 30% of patients still had a PD diagnosis at the 6-year follow-up, with no significant differences between the two treatment arms. There were also no statistically significant differences between groups concerning self-harm/suicidality, vocational functioning, and use of health care services.The findings in the present study differ from those of other step-down programs by Chiesa et al.  and FonaThe present finding of equal long-term outcomes should be considered in light of accumulating evidence of the benefits of outpatient individual therapy for patients with PDs, particularly borderline PD ,40,41. AThe present findings suggest that both hospital-based long-term step-down treatment and outpatient individual psychotherapy with therapists with the opportunity to practice long-term treatment may help improve symptoms and psychosocial in poorly functioning PD patients. Interestingly, analyses of treatment costs from baseline to the 3-year follow-up showed no significant differences in total treatment costs between the treatment conditions when both costs related to study treatment and additional psychiatric treatments were included . The preTaking into account the superior psychosocial outcome for the outpatient treatment condition at the 3- year follow-up, the divergence in the pattern of the clinical course between the treatment groups from the 3- to 6-year follow-ups was unexpected. The finding at the 6-year follow-up is strengthened by the observer-rated GAF values and self-rated WSAS scores, which both demonstrated the same statistically significant patterns between 18 months and 3 years and between 3 and 6 years. Moreover, the shift in the proportion of patients with GAF levels >60 further supports this pattern. A GAF score <60 is descriptive of moderate to severe psychosocial dysfunction, and a GAF score >60 indicates a range from mild dysfunction to a high level of functioning. At the 3-year evaluation, only one-third of patients in the step-down group and two-thirds of patients in the outpatient group had a GAF level >60 . In contSome plausible explanations concerning the divergence in clinical course between groups should be discussed. First, group therapy was an essential element in the step-down condition. Group therapy is a demanding format for patients with PDs that activates and challenges their interpersonal problems and emotional difficulties. The gains may be harder to reach and slower to appear when struggling with such core issues over time in therapy groups. However, if sufficiently integrated, the experiences might serve as a basis for further development after the end of treatment. Next, the step-down program implied a disruption of attachments to therapists and group members after a short intensive day treatment phase, and required that the patients form new emotional bonds with group members and therapists in outpatient treatment. The attachment difficulties typical of patients with severe PD make those patients vulnerable during transitions between therapies. This vulnerability may have affected their therapeutic process and caused a delay in clinical improvement . In factThe strengths of the present study are the randomized design and the extended follow-up of long-term therapies for patients with PDs. At present, both step-down models and outpatient therapies are realistic treatment options for the majority of dysfunctional PD patients, and comparisons between treatments at such different levels of care are needed. The day hospital part of the step-down program was quite representative for short-term day hospitals in Norway . MoreoveThe findings indicate that both hospital-based long-term step-down treatment and outpatient individual psychotherapy with therapists who are able to provide long-term treatment may help improve symptoms and psychosocial functioning in poorly functioning PD patients, although patients with schizotypal and antisocial PD were not included in this study. The step-down group continued to improve in social and interpersonal functioning in the post-treatment phase, suggesting that longer-term changes were stimulated during treatment. In concordance with most treatment studies of patients with PDs, there was considerable variation in outcome among the patients across treatments. We need to further improve our understanding of various patient and treatment characteristics that might influence the long-term course of these patients, and whether some treatments work better for certain patients.The authors declare that they have no competing interests.TW, SK, ØU, and GP were responsible for designing the UPP-study and selecting study instruments. MSJ, BH, and AK contributed to the acquisition of data. BTA and TW analyzed the data, with statistical contributions by OK. The manuscript was drafted by BTA and TW. EHK and BH contributed to the interpretation of results. All authors reviewed, revised, and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/14/119/prepub


Background: Glutamate system is modified by ethanol and contributes both to the euphoric and the dysphoric consequences of intoxication, but there is now growing evidence that the glutamatergic system also plays a central role in the neurobiology and treatment of mood disorders, including major depressive disorders and bipolar disorders. We speculate that, using acamprosate, patients with bipolar depression (BIP-A) can take advantage of the anti-glutamate effect of acamprosate to “survive” in treatment longer than peers suffering from non-bipolar depression (NBIP-A) after detoxification. Method: We retrospectively evaluated the efficacy of a long-term (six-month) acamprosate treatment, after alcohol detoxification, in 41 patients , who could be classified as depressed alcoholics, while taking into account the presence/absence of bipolarity. Results: During the period of observation most NBIP-A patients relapsed, whereas a majority of BIP-A patients were still in treatment at the end of their period of observation. The cumulative proportion of ‘surviving’ patients was significantly higher in BIP-A patients, but this finding was not related to gender or to other demographic or clinically investigated characteristics. The treatment time effect was significant in both subgroups. The treatment time-group effect was significant (and significantly better) for bipolar patients on account of changes in the severity of their illness. Limitations: Retrospective methodology and the lack of DSM criteria in diagnosing bipolarity. Conclusions: Bipolarity seems to be correlated with the efficacy of acamprosate treatment in inducing patients to refrain from alcohol use after detoxification (while avoiding relapses) in depressed alcoholics. Placebo-controlled clinical trials are now warranted to check the validity of this hypothesis. Glutamate is the primary excitatory neurotransmitter in the brain. Glutamatergic systems are targets for the actions of ethanol via its antagonism towards the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor and other mechanisms. The modulation of glutamatergic function by ethanol contributes both to the euphoric and the dysphoric consequences of ethanol intoxication ,2. In thActing as the major excitatory neurotransmitter in the cerebral cortex, glutamate is thought to play a role in major mental disorders, including major depressive disorders and bipolar disorders ,6,7,8,9.Although the relationship between substance abuse and bipolar disorder is complex and not yet fully understood, the idea that they could share mechanisms of self-renewal and positive mutual reinforcement has already been raised ,18,19,20We speculate that, by using acamprosate, patients with bipolar depression can take advantage of the anti-glutamate effect of acamprosate to stay in treatment for a longer time than peers with non-bipolar depression after detoxification. This hypothesis implies that acamprosate would be able to reduce the drive to drink with the outcome that their time to relapse would be longer that of peers without bipolar depression after detoxification.Total Alcohol Consumption (TAC).“relapse” to have taken place if a male patient takes more than 2 drinks on one day or if a female patient takes more than 1 drinks on one day  [Time to relapse, measured in days. We consider a alcohol) ; we do nalcohol) .Clinical Global index (CGI).Global functioning assessment (GAF).Time in days with TAC = 0.In our clinical practice, we started considering bipolarity in all alcoholic depressed patients treated with acamprosate. The main aim of the present study was to retrospectively evaluate the efficacy of a long-term (six-month) acamprosate treatment in depressed alcoholics, dividing those showing bipolarity from those without bipolarity, after alcohol detoxification, with reference to the following parameters: The secondary aim was to evaluate the correlation between demographic, clinical variables and retention in treatment.Diagnosis of alcohol dependence according to DSM-IV-R criteria .Diagnosis of major depressive episode according to DSM-IV-R criteria.Presence of multiple psychosocial or environmental problems within the previous two years.Patients consuming over 5 units of alcohol per day at treatment entry.Patients living with their families.Having successfully accomplished 7 days of a detoxification program with sodium oxybate.Having successfully recovered from a depressive state after 3 weeks, whether using or not using serotoninergic agents .Patients not treated with antiepileptics.We retrospectively considered all consecutive depressed chronic alcoholics in treatment with acamprosate at the Dual Diagnosis Unit of the Department of Psychiatry of the University of Pisa, Italy, during a three-year period, 2010–2013. The inclusion criteria were: Serious liver disorders and chronic diseases.Exclusion criteria were: The sample consisted of 41 patients, 19 (46.3%) of them males and 22 (53.7) females, average age 40.88 ± 11.5 . Patients were mostly involved in a stable sexual relationship , and were currently blue collar , with a low educational level , without welfare benefits , with adequate income , but experiencing difficulties as to social adjustment, and living in a family . According to the Hypomania Check-List (HCL) cut-off, 22 patients showed a depressive episode in the absence of bipolar spectrum (NBIP-A patients). 11 (50.0%) were males, age ranged between 26 and 38 years (average age 43.50 ± 10.9). 19 were affected by bipolar depression (HCL score ≤ 14) (BIP-A patients). 8 (42.6%) were males, age ranged between 17 and 66 years (average age 37.84 ± 11.8).Demographic data (at the beginning of treatment). We considered gender , age , education , marital status , job status , income .Bipolar spectrum. To divide our sample into patients with and without bipolar spectrum, we used the Hypomania Check-List (HCL) compiled by Angst ,28. ThisAlcohol intake (according to scheduled visits). This was evaluated in terms of units of alcohol. The easiest way to calculate this is to count the number of glasses of alcoholic drinks consumed daily, expressed as standard US drinks  .TAC = 0 (according to scheduled visits). This parameter was assessed through self-evaluation, and confirmed by family observer evaluation. One or two members of the family were responsible for detecting the intake of the medications and patient alcohol intake.Clinical Global Impressions (CGI) (monthly). Severity of illness, global improvement and efficacy index were evaluated by CGI . ClinicaSocial adjustment (monthly). This was evaluated by means of the Global Assessment of Functioning (GAF) . The GAFThe following instruments were used to collect data on the variables to be studied: A researcher who had not been informed about variations in subjects’ alcohol intake administered the CGI and GAF. There was a preference for the researcher not to be informed because CGI and GAF scales are to be rated exclusively with respect to psychological, psychopathological, social and occupational functioning.Patients were assessed monthly by the use of CGI and GAF. Patients who stayed in treatment were assessed at the end of treatment. When patients had negative outcomes, those who gave up were assessed at the time of treatment interruption, this being counted as their last regular assessment, rather than the previous month’s.Depressed patients with and without bipolarity were compared for demographic and clinical aspects by means of the chi-square test for categorical variables, and Student’s t test for continuous variables. Retention in treatment was analysed by means of the survival analysis and Wilcoxon statistics to allow the survival curves to be compared. For the purposes of this analysis, the term “terminal event” refers to patients who left the treatment after a “relapse”, while “withdrawing during interval” refers to patients who are still in treatment at the end-point, or have decided to leave treatment for reasons unrelated to the treatment itself . In other words, we consider 2 kinds of positive outcome: the first when a patient left the treatment programme with a successful result (whether because abstinent or despite a “slip”) or was referred, in the same condition, to other programmes; the second when a patient was still in treatment, at the end-point, either as an abstainer, or despite a “slip”. We consider a negative outcome to have happened if a patient has “relapsed”.Regarding clinical global impressions and social adjustment outcomes, univariate and multivariate statistical procedures have been used for cross-sectional evaluation and repeated analysis of variance for longitudinal evaluations.p < 0.05. We made use of the statistical routines of SPSS 20.0 .The statistical tests were considered significant at the level of On the basis of the demographic information that had been collected, no differences were observed, at the beginning of treatment, between BIP-A and NBIP-A patients. TAC  did not differ between BIP-A (7.74 ± 1.6) and NBIP-A (7.73 ± 1.4) patients. No differences were observed either in the CGI severity of illness , or the GAF scores .p = 0.009). None of the patients had been dismissed for being violent; none had given the treatment up because of side-effects; none had been imprisoned or hospitalized.At the end of six months, 5 (22.7%) NBIP-A and 12 (63.2%) BIP-A patients were still in treatment, while 17 (77.3%) NBIP-A and 7 (36.8%) BIP-A patients had relapsed. This difference was statistically significant .At the end of six months 8 (36.4%) NBIP-A and 9 (47.4%) BIP-A patients showed uninterrupted TAC = 0; 14 (63.6) NBIP-A and 10 (52.6%) BIP-A patients showed slips . BIP-A males showed a better retention rate (0.75) than their NBIP-A peers (0.24) . BIP-A females showed a better retention rate (0.16) than NBIP-A females (0.09) .p = 0.77). CGI improvement was assessed as “much improved” in BIP-A (1.79 ± 0.7) and NBIP-A (2.09 ± 1.3) patients . CGI efficacy index differed in BIP-A and NBIP-A patients , and time-group effect  were significant. Time-group differences were not related to the outcome . BIP-A patients (61.84 ± 10.1) and NBIP-A patients (62.73 ± 13.3) did not report significantly different degrees of social adjustment. Time effect was significant . Time-group effect  was not significant and was not related to the outcome .BIP-A patients reported a lower CGI severity of illness (2.41 ± 1.3) than NBIP-A patients (3.21 ± 1.5). Time effect , no correlation was found between retention rate and gender, age , education, marital status, job, income, severity of illness, social adjustment at study entry, or complete abstinence from alcohol.Using Cox regression life table statistics, stepwise forward  method, : paroxetine (up to 20 mg/die), sertraline (up to 150 mg/die), citalopram (up to 10 mg/die), fluvoxamine (up to 200 mg/die).In this study the following were prescribed: During the period of observation most NBIP-A patients relapsed, whereas most BIP-A patients were still in treatment at the end of the period of observation. The cumulative proportion of “surviving” patients was significantly higher in BIP-A patients and showed no correlation with gender or tother demographically or clinically investigated characteristics. Treatment time effect was significant regarding CGI and GAF in both subgroups. Considering differences between the two groups, the treatment time effect was significant (significantly better) in BIP-A patients for a single parameter: CGI severity of illness. More frequently the therapeutic effect was assessed as marked in BIP-A patients and as moderate in NBIP-A patients.At the end of our period of observation, more BIP-A patients, percentagewise, were still in treatment, whereas a higher percentage of NBIP-A patients had relapsed. In line with this observation, some studies have pointed out that bipolar alcoholics had a better outcome, as assessed by drinks per drinking day, than depressed alcoholics after a period of observation as long as 2 years , while oGender is an important variable in considering differences in the prevalence, risk, and clinical correlates of alcoholism in bipolar illness. While the prevalence of alcohol abuse or dependence is higher for bipolar men than bipolar women, the risk of developing alcoholism, by comparison with the general population is significantly higher for bipolar women ,40. DespOn the issue of endpoint evaluations, BIP-A patients reported a lower severity of illness than NBIP-A patients. Time effect and time-group effect were both significant. Moreover, time-group differences were unrelated to the outcome. These are notable findings, especially if one considers the low degree of compliance with treatments for drug addiction and for several forms of psychiatric disorder. Looking at outcome and retention in treatment, there are studies that have stressed the fact that substance use and alcohol use disorders are associated with poor clinical outcomes in patients who have a bipolar disorder ,47,48,49et al. suggest that depressive symptoms and alcohol craving both increase the proximal risks of alcohol use in individuals with co-occurring bipolar and alcohol use disorders [Acamprosate shows attractive features , but less than thrilling results: negative trials have been reported as well as large meta-analyses that support the concept that acamprosate efficacy is limited ,38,41,50isorders . In reviisorders . The effisorders ,67. We cisorders . Anti-glisorders .relapsed rather than slipped back into alcohol use).This is a retrospective study, so we can only put forward a suggestion about the possible correlation between acamprosate and a positive outcome in bipolar alcoholics after detoxification. We decided not to use DSM criteria to detect bipolarity, preferring HCL because of its higher sensitivity ,70. We cAfter detoxification from alcohol, BIP-A depressed patients stay in treatment longer than their NBIP-A depressed peers. These results highlight the fact that bipolarity seems to be significantly correlated with the outcome of acamprosate treatment, in terms of the percentage of depressed patients who refrain from alcohol use after their detoxification. Of course that placebo-controlled clinical trials are now warranted and required to check the validity of this hypothesis.


It was observed that the star-like HBPS-g-PDMAEMA formed aggregates in aqueous solution. The influence of polymer concentration, ionic strength and pH value on the aggregates in aqueous solution was investigated by using UV–vis spectroscopy and DLS analysis. The results showed that size of aggregates was affected by a corresponding stimulus. In addition, the loading ability of HBPS-g-PDMAEMA aggregates was investigated by using pyrene or Nile red as the model guest molecules by using UV–vis and fluorescence spectroscopy. The results showed that HBPS-g-PDMAEMA aggregates were capable to encapsulate small hydrophobic molecules. These newly prepared HBPS-g-PDMAEMA nanocarriers might be used in, e.g., medicine or catalysis.The topic of stimuli-responsive nanocarriers for loading guest molecules is dynamic. It has been widely studied in applications including drug controlled release, smart sensing, catalysis, and modeling. In this paper, a graft copolymer (hyperbranched polystyrene)- Stimuli-responsive polymers have attracted much attention due to their broad applications including drug controlled release –5, sensiPoly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) is a well-known stimuli-responsive polymer that responds to changes in temperature, pH and ionic strength –14. Among-PNIPAM in which HBPS was prepared by click chemistry to load small hydrophobic molecules [Hyperbranched or star-like polymers have become attractive to academia and industry in recent years due to their special chemical and physical properties. In comparison with their linear counterpart, these polymers afford promising applications such as drug delivery –17, geneolecules . Howeverolecules . It usedolecules . Produceg-PDMAEMA. HBPS segments behaved as hyperbranched topology support and also as hydrophobic segment to give HBPS-g-PDMAEMA aggregates stability in aqueous solution. The PDMAEMA segments behaved as functional chains to afford stimuli-responsive properties. The phase-transition behavior of the polymer aggregates in aqueous solution was studied. The size of the polymer aggregates under different conditions  was investigated. The loading ability for small hydrophobic molecules was studied. To the best of our knowledge, this is the first report to prepare HBPS-containing stimuli-responsive materials for loading small molecules.Here, we prepared a hyperbranched graft copolymer HBPS-g-PDMAEMA was prepared through AT-SCVP and following ATRP strategies as shown in 1H NMR spectrum of HBPS showed peaks around 5.7 and 5.2 ppm attributed to double bonds [Mn of HBPS to be 4.4 kg/mol with a molecular weight distribution of 1.85 and Mn of HBPS-g-PDMAEMA is 151.3 kg/mol with a molecular weight distribution of 2.84. The molecular weight of HBPS-g-PDMAEMA is bigger than HBPS, which demonstrated that HBPS-containing chlorine atoms successfully initiated the ATRP of DMAEMA, which resulted in HBPS-g-PDMAEMA. 1H NMR spectrum of HBPS-g-PDMAEMA. The structure of PDMAEMA was clearly confirmed according to literature [1H NMR and GPC measurements showed that the target polymer HBPS-g-PDMAEMA was successfully prepared.The target polymer HBPS-le bonds –27 as shterature  and the terature . The resg-PDMAEMA aqueous solution at a wavelength of 500 nm and a concentration of 10 mg/mL by using a UV–vis spectrophotometer equipped with a water bath, as shown in The PDMAEMA polycation becomes soluble in water due to the protonation of amine groups and hydrogen bonding with water molecules. Furthermore, PDMAEMA is sensitive to temperature, pH and ionic strength. The influence of temperature on PDMAEMA is important, because the LCST of PDMAEMA in water is near the human body temperature; a fact that might be exploited in drug delivery systems. We checked the light transmittance of the HBPS-g-PDMAEMA in water at a concentration of 10 mg/mL. g-PDMAEMA in water. When the temperature is lower than the LCST, the solution is clear, which demonstrates that the PDMAEMA segment is soluble in water. When the temperature is higher than the LCST, the solution becomes opaque, which demonstrates that the PDMAEMA chains become partially insoluble. In aqueous solution, the phase inversion depends on the formation or breakdown of hydrogen bonds between polymer chains and water molecules as well as entropically driven effects. g-PDMAEMA aggregates. It was observed that the morphology of aggregates is inhomogeneous and the aggregates were composed of clusters of smaller units. These aggregates were soluble in water due to water soluble PDMAEMA chains. The aggregates were also stable enough because the incorporated HBPS segment is an excellent hydrophobic polymeric chain.In this case, pH value and ionic strength were not adjusted and deionized water was directly used to prepare the samples. The transmittance was constant around 0.95 from 25 to 30 °C. As the temperature was increased, the transmittance was decreased and reached a stable value at 40 °C. The curve of the first derivative showed that the transmittance decreased dramatically at a temperature of about 39 °C, which is the LCST of HBPS-g-PDMAEMA aggregates in water, DLS measurements were performed and the change in aggregate size as a function of temperature and concentration were recorded. The solutions were prepared by dissolving HBPS-g-PDMAEMA in water. The results of the DLS measurements are shown in g-PDMAEMA aggregates at higher concentrations, such as 2 and 5 mg/mL, showed the maximum size at temperatures of 35–40 °C, close to the LCST of 10 mg/mL HBPS-g-PDMAEMA aggregates in water at 39 °C. Above 40 °C, the size of HBPS-g-PDMAEMA aggregates decreased with increasing temperature. The solution with concentration 1 mg/mL showed no obvious increase close to the LCST, but at higher temperatures the aggregate sizes become smaller. g-PDMAEMA. When the temperature reached the LCST, the PDMAEMA chains became insoluble in water and the relatively higher concentration made the PDMAEMA chains of two aggregates coil with each other, which led to bigger aggregates. As the temperature was increased further, PDMAEMA chains started to shrink and the size of the aggregates decreased.To further investigate the behavior of HBPS-g-PDMAEMA aggregates at NaCl concentrations of 0, 5 and 20 mM as a function of the temperature. The size of polymer aggregates decreased with increasing temperature either in deionized water or aqueous solution with relatively low concentrations of NaCl, and there was no maximum point. This is because higher concentrations of NaCl lowered the repulsive electrostatic interactions between PDMAEMA chains. So, higher concentrations of NaCl led to smaller aggregation sizes. The change of size was caused from the tendency to insolubility of PDMAEMA segments at higher temperature, but for the sample with 154 mM NaCl, the trend was very different. The size increased with heating and reached above 500 nm at 40 °C to even 2500 nm at 55 °C. The concentration of NaCl was sufficiently high to break the electrostatic interaction of PDMAEMA segments, so aggregation occurred and large sized aggregates were formed.The electrostatic interaction is a major factor to influence the behavior of PDMAEMA in aqueous solution. So in this work, the influence of ionic strength, which affects the electrostatic interactions, was studied. In this case, pH was not adjusted and deionized water was used to prepare buffer solutions of different ionic strengths. g-PDMAEMA depends on the hydrophobicity of PDMAEMA chains. The protonation of the tethered amine groups and the hydrogen bonding with water molecules are the major factors. In this work, the effect of pH on the phase-inversion behavior of HBPS-g-PDMAEMA is also investigated. In this case, the change in ionic strength was ignored and it was not adjusted by using NaCl due to the small amounts of added NaOH or HCl. The solution samples were prepared by the addition of certain amounts of polymer in aqueous solutions with certain pH values of 5.9, 7.4 and 8.5. The concentrations of all the aqueous HBPS-g-PDMAEMA solutions were 1 mg/mL. g-PDMAEMA aggregates.The phase-inversion behavior of HBPS-g-PDMAEMA aggregates was investigated by DLS analysis, the results of which are shown in g-PDMAEMA aggregates in aqueous solution. g-PDMAEMA aggregates in aqueous solution at pH 7.0, 7.4 and 8.5 at 30 and 50 °C. In deionized water, a broad size distribution of HBPS-g-PDMAEMA aggregates was obtained. The size of many aggregates in deionized water was higher than 500 nm. It is very interesting that in basic environment, the aggregates showed a lower distribution than in deionized water. The effect of temperature and pH on polydispersity index (PDI) of HBPS-g-PDMAEMA aggregates was studied, as shown in g-PDMAEMA aggregates showed a narrow distribution which means that these stimuli-responsive polymers may be suitable for medicinal use.The effect of pH on the size of HBPS-g-PDMAEMA aggregates for hydrophobic molecules, pyrene was used as a model molecule. Pyrene, a fluorescence-sensitive molecule, is often used for detecting the local polarity of amphiphiles. To prepare the pyrene/HBPS-g-PDMAEMA aqueous sample solutions, 5 to 6 mg pyrene was added in 5 mL HBPS-g-PDMAEMA solution with concentrations from 0 to 10.0 mg/mL followed by agitation for 2 h and filtration through a 0.45 μm micro-filtration membrane. g-PDMAEMA, which indicated that pyrene was transported to the aqueous solution by using HBPS-g-PDMAEMA.To confirm the loading capability of HBPS-g-PDMAEMA is an amphiphilic polymer containing a hydrophobic HBPS segment and hydrophilic PDMAEMA segments. The amphiphilic structure of this polymer affords the capability to disperse small hydrophobic molecules in aqueous solution. The ratio of I3/I1 from fluorescence spectroscopy, the intensity ratio between the peak around 382 nm and the peak around 373 nm, showed a relatively polar environment, with higher values meaning lower polarity. In this work, the pyrene/HBPS-g-PDMAEMA aqueous sample solutions were used for detecting the environmentally located polarity of encapsulated pyrene. I3  was increased to 0.68, as concentration of HBPE-g-PDMAEMA was increased to 2.0 mg/mL. Although this value is much lower than pure aliphatic hydrocarbons (1.65–1.8) [g-PDMAEMA for small hydrophobic molecules.The graft copolymer HBPE-.65–1.8) , the impg-PDMAEMA aggregates to encapsulate small hydrophobic molecules, Nile red (NR), a commonly used fluorescence dye was used as a model molecule. The preparation of HBPS-g-PDMAEMA/NR solutions was similar to HBPS-g-PDMAEMA/pyrene solutions. As shown in g-PDMAEMA aggregates can be observed by the naked eye. The solution containing these aggregates showed the pink color of NR in hydrophobic environment, and solutions without aggregates were colorless. These results indicated the successful encapsulation of NR in HBPE-g-PDMAEMA aggregates. The fluorescence spectra of HBPE-g-PDMAEMA/NR in aqueous solution shows peaks around 625 nm which were attributed to NR, as shown in g-PDMAEMA aggregates, and it was found that a large amount of NR was dispersed in water with more HBPE-g-PDMAEMA aggregates. The trend was nonlinear unlike the linear trend of NR encapsulation in micelles, as the size and aggregation of HBPE-g-PDMAEMA are influenced by the polymer concentration as shown in To further confirm the capability of HBPE-g-PDMAEMA aggregates in aqueous solution, a standard curve was drawn as shown in g-PDMAEMA/NR solutions were studied by UV–vis spectroscopy as shown in 2 is 0.99. By using the standard curve, the concentration of NR in each sample solution can be calculated. From calculation and fitting line, the result shows that 1 mg of HBPS-g-PDMAEMA could load about 28 μg NR, which is limited. It is maybe a result of low ratio of HBPS segments and this inefficient loading method. The R2 of the best linear fit line is 0.96 and it shows that the concentration of NR increasing linearly with concentration of HBPS-g-PDMAEMA. It demonstrates that HBPS-g-PDMAEMA is still intact up to a concentration of 0.1 mg/mL. Therefore, the lower critical aggregation concentration in water is beyond 0.1 mg/mL.To further investigate the loading capability for NR of HBPE-g-PDMAEMA was synthesized by ATRP and characterized by 1H NMR and GPC. The phase-inversion behavior of HBPS-g-PDMAEMA in aqueous solution was studied. Obvious optical changes were observed when the temperature reached the LCST. The influence of polymer concentration, ionic strength and pH value was also investigated. The size of the aggregates was affected by the corresponding stimulus. A higher temperature led to the shrinkage of PDMAEMA chains and resulted in the decrease in aggregates size. The polymer tends to further aggregation near the LCST at higher polymer concentrations. It was found that the basic environment results in narrow PDI of HBPS-g-PDMAEMA aggregates at a temperature above the LCST. In acidic environment, the HBPS-g-PDMAEMA aggregates showed no phase inversion. The HBPS-g-PDMAEMA aggregates/pyrene solutions were used to study the loading ability for small hydrophobic molecules. The results demonstrated that the HBPS-g-PDMAEMA aggregates were capable of encapsulating small hydrophobic molecules. The I3/I1 value reached 0.7 when the polymer concentration was above 2.0 mg/mL. The encapsulation of Nile red showed that HBPS-g-PDMAEMA aggregates are good containers for small hydrophobic molecules.In this paper, the graft copolymer HBPS-N,N,N′,N″,N″-pentamethyldimethylenetriamine (PMDETA) were used as received.All reactions were performed under argon and argon gas was purified by passing through four columns loaded with potassium hydroxide, 4 Å molecular sieves, silver molecular sieves and potassium/sodium alloy. Vinylbenzyl chloride (VBC) was purified by passing through a column packed with alkaline alumina. DMAEMA and benzyl chloride (BC) were used as received. Water was deionized before use. CuBr was purified by using acetic acid and methanol before use. Alkaline alumina, CuCl, 2,2-bipyrene (bpy) and g-PDMAEMA are shown in The synthetic routes to HBPS and HBPS-g-PDMAEMA was synthesized by using a Schlenk line system under argon atmosphere. The solution of Cu complex was prepared by dissolving 76.1 mg CuBr and 121.0 μL PMDETA in 7 mL anisole which was then deoxygenized by three freezing–thawing–freezing cycles. The initiator solution was prepared by dissolving 36.1 mg HBPS in 4.0 mL anisole. The monomer solution was prepared by dissolving 2.5 mL DMAEMA (14.9 mmol) and 1 mL of initiator solution containing 9.0 mg HBPS  in 3.0 mL anisole. 1.0 mL Cu complex solution containing 10.9 mg CuCl (75 μmol) and 17.3 μL PMDETA (82 μmol) were added into monomer solution. The reaction mixture was heated to 90 °C in an oil bath under constant stirring. After 3 h, the polymerization was stopped by immersing the reaction mixture in liquid nitrogen and putting it under ventilation. The product was precipitated by dropwise addition of the reaction solution in a large amount of hexane. After filtration, the white product was collected and then redissolved in THF. In order to remove the Cu complex, the diluted solution was passed through a neutral alumina column. The eluted solution was concentrated and dropwise added in large amount of hexane to precipitate the product. This process was repeated and then the obtained product was dried under vacuum at 40 °C.HBPS-1H NMR) spectra were recorded on a Bruker 400M spectrometer. The molecular weight of polymer was determined by gel permeation chromatography (GPC) (Waters-Wyatt) equipped with RI, UV, viscosity and LS detectors using tetrahydrofuran (THF) as eluent. The UV–vis spectra were recorded on a UV3802 (UNICO) ultraviolet spectrophotometer. The size and zeta potential of the polymer in aqueous solution at different temperature were measured on a Zetasizer 3000HSA dynamic light scattering (DLS)/zeta potential analyzer. The fluorescence spectra were recorded on an F-280 (Tianjin Gangdong) fluorescence spectrophotometer.The proton nuclear magnetic resonance (


Drosophila melanogaster DExH box/Tudor domain protein Spindle-E, whose activity is necessary for the generation of most germline piRNAs. In this study we molecularly and phenotypically characterized 14 previously identified spindle-E alleles. Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain. Additionally, we found that processes that depend on piRNA function, including Aubergine localization, Dynein motor movement, and retrotransposon silencing, were severely disrupted in alleles with DExH box domain mutations. The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much. From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.Transposable selfish genetic elements have the potential to cause debilitating mutations as they replicate and reinsert within the genome. Therefore, it is critical to keep the cellular levels of these elements low. This is especially true in the germline where these mutations could affect the viability of the next generation. A class of small noncoding RNAs, the Piwi-associated RNAs, is responsible for silencing transposable elements in the germline of most organisms. Several proteins have been identified as playing essential roles in piRNA generation and transposon silencing. However, for the most part their function in piRNA generation is currently unknown. One of these proteins is the  A large portion of both the human and Drosophila genomes are composed of transposable elements (TEs), which are capable of creating genome instability and a high mutation rate on excision and re-integration within the genome , are responsible for silencing TE expression and transposition   . Most prne (Aub) . Other ppiwi mutant cells ; FRT82B UbiGFP male flies (Bloomington stock center). Second or third instar larvae were heat-shocked at 37° for 2 hr on two consecutive days to induce clones. Female flies of the correct genotype were dissected 10 d after heat shock.Germline clones were induced using the FLP/FRT system . FRT82B mutant/spn-E∆125spn-E flies as in . Multiple sets of spn-E gene-specific primers that span the gene from the start codon to the stop codon, including introns, were used (primer sequences available upon request). PCR products were purified using the Qiagen MinElute PCR purification kit. Sanger sequencing was performed using the Big Dye termination kit.Genomic DNA was isolated from 30 adult male es as in . spn-E∆1n-E gene . The spnRabbit polyclonal antisera directed against peptide MNLPPNPVIARGRGRG (amino acids 1–16) . Ovaries were homogenized five- to 10-times in lysis buffer . Samples were centrifuged twice for 5 min at 12,000g at 4° and the supernatant was assayed for protein concentration. Protein was quantitated by a Bradford-type assay using the Biorad Protein Assay kit; 40 μg of protein was resolved on 7% SDS-polyacrylamide gels and transferred to PVDF membranes (Immobilon) using the Bio-Rad mini-PROTEAN tetra electrophoresis system. Western blots were performed as described previously spn-E were placed on yeast overnight, put into egg-laying chambers, and allowed to lay eggs on apple juice agar plates with yeast paste overnight at 25°. Eggs of each class were counted after 24 hr of egg-laying for three consecutive days.Eight to 10 female flies that were 2 or 3 days old of the specified genotypes  that were 2 or 3 days old were placed on yeast overnight. Ovaries were dissected in EBR and placed in microcentrifuge tubes. The EBR was removed and the ovaries were flash-frozen in liquid nitrogen or used fresh for RNA isolation. RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was treated twice with Turbo DNase (Ambion) to remove contaminating genomic DNA. cDNA was generated from 1 µg of RNA using the Verso cDNA kit (Thermo Scientific) or the Maxima cDNA kit (Fermentas). A 10-µl real-time PCR reaction was performed with either 1× ABsolute Blue SYBR Green master mix (Abgene), 0.075 mM of forward and reverse primers, and 1 µl of cDNA reaction or 1× Maxima SYBR master mix (Fermentas), 0.3 mM of forward and reverse primers, and 1 µl of cDNA reaction. Cycling parameters were: 50°, 2 min; 95°, 10 min; 95°, 15 sec; and 60°, 1 min for 40 cycles using an ABI 7900HT. The following previously published primers were used: HetA and TART as described elsewhere  as well as two additional spn-E alleles that had been previously phenotypically characterized (616 and 653) .We obtained 12  protein    do not espn-E gene contained mutations that lie in the known functional domains: the DExH box; the Tudor; or the Zinc finger , a common motif found in proteins that bind and hydrolyze NTPs. Motif II , which is necessary for ATP binding/hydrolysis and interdomain contacts, and motif V (8D4-11S435F), which may be necessary for RNA binding. In addition, we identified a mutation that is between motifs I and II in an amino acid that is conserved between species (7G2-5T233I)  . The ide2His2-like fold group that is identified by the sequence, X2-Cys-X2,4-Cys-X12-His-X3,4,5-His, where the two Cys and two His amino acids are important for coordinating Zn  eggshell patterning, AUB subcellular localization, and dynein motor complex localization. Additionally, we directly tested whether the piRNA pathway was affected in the mutant ovaries by measuring retrotransposon levels .spn-E mutant females lay eggs with severe D/V patterning defects ranging from fused dorsal appendages to no dorsal appendages and collapsed eggs , which lies in DExH box motif I, lay approximately 94% collapsed eggs and no wild-type eggs. This is in contrast to wild-type females that do not lay collapsed eggs , but compares favorably with eggs laid by spn-E mutant females that do not express detectable SPN-E protein, which lay between 83% and 100% collapsed eggs . The same severe phenotype occurs in eggs laid by spn-E mutant females that have mutations located in DExH box motif V (8D4-11S435F) and in between motifs I and II (7G2-5T233I).sed eggs . To eval defects . In contE239K, 23-17H241Q). Mutation of the Glu in this region leads to a less severe phenotype than the DExH box mutations discussed above, where the majority of eggs laid are collapsed but a larger percentage of eggs with fused or no dorsal appendages are present. Additionally, mutation of the His in the DExH box (23-17H241Q) sequence resulted in an even less severe phenotype with 55% of the eggs laid having wild-type patterning.We found two mutations in amino acids in the DExH box motif, region II (155-55P508L), which changes a conserved Pro to Leu in between the DExH box and the Tudor domains, produced a mild D/V patterning defect with 60% of the eggs laid having a wild-type appearance  lay eggs with an extremely mild D/V patterning phenotype, resulting in 92% of the eggs having a wild-type appearance.Flies expressing SPN-E with a mutation in the Zn finger motif . We also did not detect protein of the correct size for SPN-E by Western blotting. Nevertheless, the weaker mutant phenotype indicates that at least some functional protein may be made by this allele. Along these same lines, eggs laid by females expressing genetically characterized spn-E hypomorphic alleles, 616spn-E, and 653spn-E . It is possible that 616spn-E, 653spn-E, and 100-37spn-E express protein that we are unable to detect by our assay.Interestingly, eggs laid by females from lines 100-37spn-E653  show a sspn-E mutant ovaries to the same extent as the D/V phenotypes described above, with those alleles that show the most severe D/V patterning defects having the lowest GRK levels . Additionally, we did not detect any Grk expression in the stronger spn-E alleles, 2A9T145I, 7G2T233I, 8D4S435F, 114-33R918*, 9A9W632*, or 4E2R695*. For the 155-55E239K, 100-37splicesite488, 616R1081*, and 653Y636* alleles, which have moderate D/V patterning defects, we did not detect significant Grk levels at the dorsal-anterior corner of the oocyte at stage 9 and beyond; however, we did detect reduced GRK in the oocyte at the earlier stages . Therefore, it is possible that some GRK is expressed and localized at the later oogenesis stages in these alleles, but the level is not detectable by our immunofluorescent assay.To further analyze the D/V phenotype, we examined the expression/localization of the dorsal determinant Gurken (GRK) by immunohistochemistry. At oogenesis stage 8, GRK localizes to the dorsal-anterior corner of the oocyte, where it is necessary to signal to the dorsal follicle cells to establish embryonic dorsal fate. In support of the above data, we found that GRK levels at the dorsal corner of the oocyte are reduced in the From the above data, it seems that the DExH box domain of SPN-E is quite important for embryonic axis specification. However, the Zn finger seems to be dispensable.spn-E mutant ovaries using immunohistochemistry. To compare mutant and wild-type expression/localization in the same ovary, we generated clones of homozygous mutant egg chambers in an otherwise heterozygous background using the FLP/FRT technique . We found results similar to those shown above, with the exception of the 4-48E717K allele. The 4-48 hemizygous mutant egg chambers showed a little more punctate AUB localization than the clones; however, AUB was still localized to a certain extent. Therefore, similar to what we saw with D/V patterning, ovaries expressing SPN-E with mutations in the DExH box domain give a similar phenotype to the strongest spn-E mutant phenotype, with AUB delocalized from the nuage, demonstrating the importance of the DExH box domain to SPN-E function.AUB and SPN-E co-localize to the nuage in wild-type ovaries and AUB localization depends on SPN-E function . Therefoechnique . Similarild-type . In ovarild-type . The AUBspn-E mutants and determined whether dynein aggregates form by immunohistochemistry using EGL as a marker for the aggregates. Similar to what we found with the D/V patterning phenotype, those spn-E mutant flies that lay eggs with the most severe D/V patterning defects formed dynein motor complex aggregates .In wild-type egg chambers, dynein motor complex proteins are transported from the nurse cells to the oocyte, ultimately leading to a high concentration of protein in the oocyte and a more diffuse pattern in the supporting nurse cells . This isgregates , whereasgregates . Additiogregates . The faiDrosophila ovary . The germline-specific retrotransposons included the non-LTR retrotransposons, HetA, TART, and I Factor, and the LTR retrotransposons, Blood and roo, whereas the somatic retrotransposon used was gypsy. We found a similar trend in phenotypic severity for germline retrotransposon RNA expression in the spn-E mutant ovaries as we found for the D/V patterning defects, AUB localization, and dynein aggregate formation that we described above. Ovaries from flies expressing SPN-E protein with mutations in the DExH box region had the highest ovarian retrotransposon RNA levels. However, mutations outside of the DExH box region, including the mutation in the Zn finger motif, produced a more moderate phenotype. Interestingly, all alleles showed increased levels of the Blood and HetA retrotransposons to some extent; however, those spn-E mutants that gave the strongest developmental phenotypes showed considerably higher levels of HetA and Blood compared with the other alleles. For example, 155spn-E-55(E239K) ovaries had ∼500× the level of Blood and ∼200× the level of HetA retrotransposon RNA as heterozygous controls, whereas 66-21(H1422L)spn-E ovaries had only ∼40× the level of Blood and ∼50× the level of HetA as the heterozygotes. Additionally, only those alleles that gave the strongest developmental phenotypes also had high levels of I Factor, TART, and roo. SPN-E has previously been reported to function only in the ovarian germline cells; therefore, we did not expect to see an effect on the somatic retrotransposon gypsy  showed that an amino acid with –OH group at the analogous position in the NPH-I DExH box is necessary for its ATPase activity , a Cytoplasmic Polyadenylation Element binding protein that functions in translational regulation, mutant ovaries also show premature cytoplasmic streaming similar to what is seen in spn-E mutant egg chambers (grk RNA mislocalization that, in turn, would lead to embryonic patterning defects. Orb levels are reduced in spn-E mutant ovaries; therefore, it is possible that Orb may be either a direct or an indirect target of SPN-E function (The one exception to the above statement is I-R hybrid dysgenic crosses where the only retrotransposon that is upregulated is I Factor, the DNA damage checkpoint is not activated, yet moderate D/V patterning defects are seen in the eggs laid by the I-R dysgenic females (4-48spn-E eggs. Additionally, the retrotransposon that is most affected in 4-48spn-E ovaries is I Factor. The D/V defects that arise in eggs laid by dysgenic females are thought to arise from a competition of I Factor and grk RNA for access to the microtubule motor machinery, which leads to the displacement of grk RNA and the resulting D/V patterning defects (4-48spn-E ovaries and why I Factor levels are the most impacted by the 4-48spn-E mutation.Additionally, it has been shown that in spn-E mutants that have only a slight increase in retrotransposon levels, dynein aggregates do not form. This is also true in I-R dysgenic ovaries (spn-E mutant ovaries (In addition to D/V patterning and checkpoint activation, it seems that dynein aggregate formation is also sensitive to retrotransposon levels. We found that in R1081* allele has a mutation that causes a premature stop after the Tudor domain, whereas the rest of the alleles with premature stop codons would cause protein truncation before the Tudor domain. This allele has a weaker D/V patterning phenotype than these other alleles, indicating that if this allele does produce protein, then the Tudor domain may be an important functional domain of SPN-E. A more directed mutagenesis approach may be necessary to determine the relevance of the Tudor domain for SPN-E function.Our experiments have identified several mutations in the DExH box helicase domain of SPN-E implicating helicase function as important for SPN-E function, especially in the piRNA pathway. Although these mutations could affect protein folding, we think that the mutations we have identified affect protein function rather than protein structure because the ovarian protein levels for several of these new alleles were close to wild-type. It is interesting to note that we did not obtain mutations in the highly conserved Tudor domain. It is possible that mutations in the Tudor domain could render SPN-E unstable. However, it is curious that the 616


Meloidogyne are biotrophic plant parasites that exhibit different life cycles and reproduction modes, ranging from classical amphimixis to obligatory mitotic parthenogenesis (apomixis), depending on the species. Meloidogyne incognita, an apomictic species, exhibits a worldwide distribution and a wide host range affecting more than 3000 plant species. Furthermore, evidences suggest that apomixis does not prevent M. incognita from adapting to its environment in contrast to what is expected from mitotic parthenogenesis that should theoretically produce clonal progenies. This raises questions about mechanisms of genome plasticity leading to genetic variation and adaptive evolution in apomictic animals. We reasoned that epigenetic mechanisms might in part be responsible for the generation of phenotypic variants that provide potential for rapid adaptation. We established therefore a pipeline to investigate the principal carriers of epigenetic information, DNA methylation and post-translational histone modifications. Even if M. incognita possesses the epigenetic machinery i.e., chromatin modifying enzymes, 5-methyl-cytosine and 5-hydroxy-methyl-cytosine content is absent or very weak. In contrast, we demonstrated that the canonical histone modifications are present and chromatin shows typical nucleosome structure. This work is the first characterization of carriers of epigenetic information in M. incognita and constitutes a preamble to further investigate if M. incognita development and its adaptation to plant hosts are under epigenetic control. Our pipeline should allow performing similar types of studies in any non-model organism.Root-knot nematodes of the genus  Epigenetics is the study of heritable changes in gene expression and function that cannot be explained by changes in DNA sequence. These molecular mechanisms can be influenced by the environment and the individual history as well as being potentially transferable from a generation to the next, with a reversible character  of the genus Meloidogyne are obligatory plant parasites that constitute major agricultural pests worldwide. They infect almost all cultivated plants and establish an intimate interaction with their hosts inducing the re-differentiation of root cells into hypertrophied and multinucleate feeding cells  and 5-hydroxy methylcytosine (5-hmC) in genomic DNA by LC-MS/MS. We then used Western blots to verify the presence of histone and histone modifications and tested critical parameters of a standardized N-ChIP procedure that need to be adapted for each model.Many non-model organisms of ecological and economical importance such as DNA methylation is a common feature of many but not all genomes. We recommend starting with this feature.M. incognita possesses the capacity to methylate DNA, we first conducted reciprocal BLASTp searches combined to OrthoMCL analysis to identify proteins that might be related to known chromatin-remodeling enzyme orthologs . Animal DNA methyltransferases can be subdivided into three subfamilies, DNMT-1, DNMT-2 and DNMT-3, based on sequence similarity  was performed with DNMT-1, DNMT-2, DNMT-3 protein sequences from nematodes ; P. pacificus Ppa- DNMT-2 (accession number AY766101.1) and T. spiralis DNMT-22 (accession number EFV60295.1); T. spiralis DNMT-3 (accession number EFV54759.1). As for the 11 other nematodes in which DNA methylation has been studied, with exception of T. spiralis  proteins, which encodes another essential component of the methylation system , C. briggsae  and P. pacificus  all contain an mbd-2-like gene  of M. incognita that were downloaded from NCBI-dbEST public database , filtered by size (≥500 bp) leading to 41,649 sequences of which 5000 were randomly chosen for CpG observed/expected ratio (CpGo/e) analysis. Most of the data came from M. incognita strain Morelos maintained at INRA Sophia Antipolis and recently published , the model nematode lacking DNA methylation process, D. melanogaster  and on A. mellifera . D. melanogaster has been previously described for its lack of DNA methylation at late stages of development  as expected for species lacking such process . However, careful analysis of the curve fitting suggests the presence of two populations of sequences: one with a mean value around 0.9 and another with a mean value around 0.7. These results could indicate the presence of two populations of genes in M. incognita: one with no methylation and one with very low methylation.To evaluate the degree of DNA methylation, we calculated the empirical distribution of CpGo/e ratios for s Figure . A simils Figure . This pae Figure . In M. iM. incognita eggs with the CTAB method . As previously described by Rosso et al., In the absence of DNA methylation, PTMs could represent one of the primary epigenetic transcriptional control mechanisms. Such a process has been shown to play a key role in the regulation of gene transcription of parasites, e.g., the apicomplexan family of protozoa and the metazoan M. incognita DNA, we tested 12 commercial antibodies, directed against histone H3 and several H3 and H4 modifications, on M. incognita J2s with western blots.The nucleosome is subject to a dizzying array of posttranslational modifications, which work alone or in combination to constitute a histone code that regulates chromatin structure and function  by the semi-dry method (SEMI-PHOR Bio-Rad). The membrane was blocked overnight at 4°C in blocking buffer and incubated with one of the 12 following commercial antibodies (1μg/μl): Anti-histone H3 ; Anti-Histone H3 dimethylated at lysine 4  or at lysine 36 ; Anti-Histone H3 trimethylated at lysine 4 , at lysine 9  or at lysine 27 ; Anti-Histone H3 acetylated at lysine 9 ; Anti-Histone H4 trimethylated at lysine 20  and Anti-hyperacetylated Histone H4 . Bands were revealed by Enhanced Chemical Luminescence (ECL Pierce) and direct exposure to X-ray film (Amersham).Western blots were performed as previously described by Azzi et al. . BrieflyThe antibodies should give a unique staining on the membrane (one unique band) to be selected for DNA immuno-precipitation. Three antibodies, Anti-H3K4Me3 (Abcam), Anti-H3K9Me3 (Abcam and Upstate), did not give any signal  digestion of chromatin to reveal its nucleosome structure, which is the fundamental unit of chromatin. Because DNA portions of nucleosome core particles are less accessible for MNase than linking sections, DNA gets digested into fragments of lengths equal to multiplicity of distance between mono-nucleosomes. First, to access nuclei, we tested different grinding times, from 3 to 10 min, for cell lysis in Dounce homogenizer. As a measure for grinding efficiency, we colored nuclei using Hoechst, a fluorescent stain that binds strongly DNA. Also, because M. incognita life-stages are really different in shape and composition, we tested these grinding conditions on both J2s and eggs. Best results were obtained with 10 min total in Dounce homogenizer, distributed as 5 min grinding following by 5 min at rest  concentrations and incubation times (see Results). Nuclei were stained with Hoechst 33342 fluorescent stain  and observed under microscope  before and after grinding.To immunoprecipitate u et al. . Each stM. incognita with increasing antibody volume ranging from 0 to 16 μl (1 μg/μl), for each of 3 tested antibodies (among the 6 antibodies that could be use): Anti-H3 (Abcam), Anti-H3K4Me3 (Millipore), and Anti-H4K20Me3. For each antibody concentration, DNA from the bound and unbound fractions was extracted with phenol/chloroform so we could quantify the sequences associated with each antibody by qPCR. In other species, histone marks such as H3K4Me3 were enriched around the transcription start sites (TSSs) of genes and were associated with active transcription , 95°C for 10 s, 62°C for 5 s, 72°C for 16 s; melting curve, 60–95°C with a heating rate of 0.11°C/s and continuous fluorescence measurement, and a cooling step to 40°C. For each reaction, the cycle threshold (Ct) was determined using the “2nd derivative” method of the LightCycler 480 Software release 1.5. Primers were designed for this experiment with the Primer3 web-interface ; Melting curve and negative first derivative of the melting-curve were recorded for each qPCR product to validate that only one product was amplified (one peak was observed).Because the antibody should be in excess over the histone of interest for ChIP, we tested protein/chromatin precipitation efficiency in The amount of target DNA recovered in the immune-precipitated fraction was quantified by calculating the percent input recovery (% IR) normalized with the percent input recovery obtained with the housekeeping gene as previously described by Cosseau et al. .M. incognita mono-nucleosome length. With M. incognita DNA, antibodies Anti-H3 and Anti-H4K20Me3 never reached saturation up to 16 μl , to obtain fragmented chromatin into mono-nucleosome to penta-nucleosomes, a size range that is optimal for ChIP, was 5min. Finally, qPCR experiments indicated that N-Chip procedure should be performed with an excess of 8 μl antibody Anti- H3K4Me3. Now that chromatin immunoprecipitation has been optimized in M. incognita it opens new perspectives to further investigate the genome-wide distribution of PTMs by high-throughput sequencing (N-ChIP-Seq).To summarize, we optimized the ChIP protocol for C. elegans. Moreover, functional DNA methylation has been recently reported in the parasitic nematode T. spiralis  and lysine 9 .In addition, it is to be noted that nematodes could be divided in monocentric species and holocentric species (Subirana and Messeguer, M. incognita constitutes an ideal model to study these mechanisms, especially as inheritance of the nematode virulence does not segregate in accordance with Mendel's laws together with asexual multiplication in host plants (Castagnone-Sereno et al., M. incognita provides support for the study of epigenetic heredity distinct from the canonical Mendelian rules. Recently, it became apparent that parasitism success is partly accomplished by epigenetic means in several parasites, including, among others, protozoans Plasmodium and trematodes Schistosoma. More especially, PTMs plays a key role in the regulation of gene transcription of these parasites (Mourão et al., Plasmodium falciparum (Freitas-Junior et al., Schistosoma mansoni, N-ChIP-seq experiments have been successfully performed to compare chromatin structure at different stages of the parasite life cycle, on different strains and on different sexes (Caby and Pierce, S. mansoni epigenetic regulation have lead to the identification of promising new targets for the development of new drug to combat schistosomiasis (Pierce et al., One major role for epigenetic variation in evolution would be to promote phenotypic variability, and allow populations to widely explore new environmental conditions. However, the detailed mechanisms thanks to which parasites evade host immunity for instance are poorly understood. M. incognita make the study of epigenetic machinery possible and susceptible to break new ground.The importance of PTMs in regulating crucial developmental processes in parasites combined with the recent resolution of the genome sequences of M. incognita, this study details DNA methylation and nucleosome structure, carriers of epigenetic information. Genomic DNA of M. incognita is not methylated. The general characteristics of nucleosome structure that we observed in the M. incognita genome are in accordance to what epigenetic studies have reported from other invertebrate species. Histone modifications, important markers of function and chromatin state, were identified in M. incognita. This study opens the way for analyzing epigenetic mechanisms in M. incognita at a whole-genome scale to identify new biological processes involved in the generation of phenotypic variation in absence of sexual reproduction in an ecological and economical important model.For the first time in Conceived and designed the experiments: Céline Cosseau, Christoph Grunau, Laetitia Perfus-Barbeoch, Pierre Abad, Philippe Castagnone-Sereno. Performed the experiments: David Roquis, Laetitia Perfus-Barbeoch, Loris Pratx, Michael Reichelt, Sara Fneich. Analyzed the data: Céline Cosseau, Christoph Grunau, Laetitia Perfus-Barbeoch, Pierre Abad, Philippe Castagnone-Sereno. Wrote the paper: Céline Cosseau, Christoph Grunau, David Roquis, Laetitia Perfus-Barbeoch, Michael Reichelt, Pierre Abad, Philippe Castagnone-Sereno, Sara Fneich.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present study is to investigate the brain activation associated with the informative value of negative feedback in rule acquisition. In each trial of a segmented Wisconsin Card Sorting Test, participants were provided with three reference cards and one target card, and were asked to match one of three reference cards to the target card based on a classification rule. Participants received feedback after each match. Participants would acquire the rule after one negative feedback (1-NF condition) or two successive negative feedbacks (2-NF condition). The functional magnetic resonance imaging (fMRI) results indicated that lateral prefrontal-to-parietal cortices were more active in the 2-NF condition than in the 1-NF condition. The activation in the right lateral prefrontal cortex and left posterior parietal cortex increased gradually with the amount of negative feedback. These results demonstrate that the informative value of negative feedback in rule acquisition might be modulated by the lateral prefronto-parietal loop. The Wisconsin Card Sorting Test (WCST) is one of the most widely used neurocognitive measures to evaluate cognitive flexibility, problem-solving, rule learning, as well as brain disorders –12. DuriThe feedback has two main values: valence and informative value , 14. TheHowever, Monchi et al.  did not We developed a segmented WCST to study the brain activation associated with NF in rule acquisition. The stimulus was similar to the standard version of the WCST. In each trial, one target card and three reference cards were displayed. Each reference card shared only one perceptual attribute  with the target card. Participants were required to choose a reference card that was the same class with the target card. Feedback was presented after each match. Before the task, participants were informed that the classification rule might pertain to one of three perceptual attributes of stimulus. If participants received NF at the first match, the excluded rule at the first match was not allowed to choose at the second and third matches. Otherwise, no feedback was displayed on the screen. As a result, they would acquire the rule after two attempts at most and they must match correctly at the third attempt. Accordingly, participants’ rule acquisition behavior can be classified into three conditions. In the 0-NF condition, participants luckily acquired the rule at the first match, so they did not receive any NF in this condition. In the 1-NF condition, participants failed at the first match, but responded correctly at the second and third match, so they receive one NF in this condition. In the 2-NF condition, participants failed at the first and second match, but responded correctly at the third match, so they receive two NFs in this condition.The difference between the 2-NF condition and 1-NF condition is the amount of NF. We compared the difference in brain activation evoked by the 2-NF condition with that of the 1-NF condition. Because the crucial function of NF in rule acquisition is to guide the participants to rule out the invalid hypothesis and turn to searching for a new hypothesis , 25, 27,Sixteen healthy right-handed volunteers  participated in this experiment. All participants met the criteria for magnetic resonance imaging (MRI) scanning . All participants reported normal or corrected-to-normal vision and the absence of neurological or psychiatric impairments. This study has been approved by the Institutional Review Board at Liaoning Normal University. We had obtained appropriate ethics committee approval for the research reported, and all subjects gave written informed consent in our experiment.A segmented WCST modified from the standard version of the WCST task was used , 7. One Each trial started with a fixation cross. A new target card was then presented on the first screen. The participant chose one of the three reference cards by pressing a button for 1, 2, or 3. The length of each match period depended on the participant’s response time, but the allowed maximum response time was 6000 ms. If the participant did not responded within 6000 ms, “No response” would appear on the screen. After the response, the feedback of was displayed, remaining on the screen for 1000 ms. Then, the target and the new reference cards would appear on the second and third screens, and participants would respond with new match. The rule was constant for one trial, and was changed between trials . Before Depending on the participants’ response and feedback, three conditions were defined. In the 2-NF condition, participants received two NFs. In the 1-NF condition, participants received a NF after the first match. At the second and third match, they responded correctly. In the 0-NF condition, they luckily gave the correct responses for all of the three matches. There were five runs.Each run had 3 conditions and 18 trials. Each condition had 6 trials in one run. A total of 90 trials were equally assigned to the three conditions, which were executed by the E-prime software. Because each condition has the same number of trials in each run, the possibility of participants receiving negative feedback and positive feedback is 2/3 and 1/3, respectively, at the first occasion. If participants received negative feedback at the first occasion, the possibility of receiving negative feedback and positive feedback at the second occasion is 1/2 and 1/2, respectively. If participants received positive feedback at the first occasion, the possibility of receiving negative feedback and positive feedback at the second occasion is 0 and 1, respectively.hwww.pstnet.com). Responses were obtained using a magnet-compatible response system.Scanning was performed with a standard whole-head coil using a 3.0 T full-body magnetic resonance imaging scanner . The blood oxygenation level-dependent (BOLD) contrast was acquired using echo planar T2*-weighted imaging. The parameters used were: repetition time = 2000 ms, echo time = 30 ms, field of view = 220 × 220 mm, flip angle = 90°, matrix size 64×64, 32slices, slice thickness = 3mm (0.99 mm gap), in-plane resolution = 3.43 × 3.43 mm, 246 volumes per run. The stimulus was presented using a magnet-compatible projector that back-projected visual images onto a screen mounted above the participant's head. The experimental task was programmed using E-Prime package . The first two volumes in each session were excluded. Images were slice-time corrected, motion corrected, re-sampled to 3 × 3 × 3 mm isotropic voxels, normalized to the Montreal Neurological Institute (MNI) space, and spatially smoothed using an 8 mm full width at half maximum isotropic Gaussian kernel. After preprocessing, participants whose head motion exceeded 3.0 mm and 3.0° were excluded from the study. At the first level, the time series in each voxel was high-pass filtered to 1/128 Hz and modeled for temporal autocorrelation across scans with an AR (1) model. The first level  statistical analysis was conducted with the general linear model to form statistical parametric maps of the Paired T-statistic. The contrasts were as follows: 2-NF vs 1-NF, 2-NF vs 0-NF, and 1-NF vs 0-NF. Individual contrast images were entered into a second level analysis to make inferences at the group level. In order to assess areas associated with NF function , a conjunction analysis (2-NF vs 0-NF ∩ 1-NF vs 0-NF) was conducted. A FDR corrected of ROI analyses were performed using the Marsbar toolbox in SPM8 . The defp < 0.05), while the right putamen reached a lower level of significance .In comparison with the 1-NF condition, the 2-NF condition activated the bilateral inferior frontal Gyrus (IFG), bilateral DLPFC, bilateral posterior parietal cortex (PPC), and right putamen . ActivatCompared with the 0-NF condition, the 2-NF condition activated the left supplementary motor area (SMA), bilateral PFC, bilateral PPC, left inferior temporal Gyrus, right fusiform Gyrus, left lingual gyrus, bilateral calcarine fissure, right caudate body, left thalamus, and bilateral declive. In comparison with the 0-NF condition, the 1-NF condition activated the right SMA, left PFC, bilateral PPC, left middle occipital gyrus, right lingual gyrus, right insula, left caudate body, and left declive. To further reveal the differences between the two NF conditions and 0-NF condition, we performed a conjunction analysis  of 2-NF .The ROI analyses were performed for ten defined regions, which were selected based on Monchi al et. . Two of F = 11.4, p < 0.01. Pairwise comparisons with Bonferroni correction demonstrated that more activation was observed in the 2-NF condition than in the 1-NF condition (p< 0.05). The activation in the right VLPFC was also larger in the 1-NF than in the 0-NF condition (p< 0.05). For the left VLPFC, a main effect of condition was not found.The analysis for the right VLPFC revealed a main effect of condition, F = 15.3, p < 0.001.Pairwise comparisons with Bonferroni correction revealed that more activation was observed in the 2-NF condition than in the 1-NF condition (p < 0.05). The activation in the right DLPFC was also larger in the 1-NF condition than in the 0-NF condition (p < 0.01). For the left DLPFC, a main effect of condition was not found.The analysis for the right DLPFC revealed a main effect of condition, F = 10.8, p < 0.01. Pairwise comparisons with Bonferroni correction revealed that more activation was seen in the 2-NF condition than in the 1-NF condition (p < 0.05). The activation in the right ACC was also larger in the 1-NF condition than in the 0-NF condition (p <0.05). The analysis for the left ACC revealed a main effect of condition, F = 10.7, p < 0.005. Pairwise comparisons with Bonferroni correction indicated that there was more activation in the 1-NF condition than in the 0-NF condition (p <0.05).The analysis for the right ACC revealed a main effect of condition, F = 13.3, p < 0.01. Pairwise comparisons with Bonferroni correction demonstrated that more activation was seen in the 2-NF condition than in the 1-NF condition (p < 0.05). The activation in the left PPC was also larger in the 1-NF condition than in the 0-NF condition (p< 0.05). For the right PPC, a main effect of condition did not reach statistical significance. The analysis for the bilateral caudate nucleus did not reach the significance in the main effect.The analysis for the left PPC revealed a main effect of condition, Previous studies have investigated the cognitive function and related brain activation of NF in the WCST. However, the functions of NF in the different stages of the WCST were not distinguished from each other , 27. We The three conditions differ in the amount of NF. Previous studies have demonstrated that feedback has two values in a rule-learning task: valence and informative value , 14. In The VLPFC has been demonstrated to be active when participants receive NF , 39. In The activation of the DLPFC, especially the right DLPFC, increased with the amount of NF. Previous studies have revealed that the DLPFC is involved in rule searching, rule selection, and information updating , 41–44. In the WCST, NF activated the PFC-basal ganglia circuits . The actIn addition, compared with the 0-NF condition, the 1-NF and 2-NF conditions activated not only the LPFC, but also the left SMA. The SMA was possibly recruited in searching for the new hypothesis, being primarily responsible for preparing a rule-based action response. That is, the SMA might form a new connection between the stimulus perceptual attribute of the stimulus and a rule-based response , 46.Finally, we found that compared with the 0-NF condition, the NF conditions activated the bilateral insula. Previous studies indicated that the insula is related to negative events , 48. TheIn conclusion, a segmented WCST was used to study the brain activation associated with the informative value of NF in rule acquisition. The activation of the LPFC including the VLPFC and DLPFC may reflect the informative value of NF. The activation of the insula may reflect the valence of NF related to the emotional experience that occurred after the failure of rule acquisition. These results expand our understanding about the different functions of NF in rule learning.